RNAi therapeutics have been regarded as a potentially powerful way to treat various genetic diseases using chemically modified siRNAs;however, current capabilities for designing effective siRNAs to target specific diseases is limited since little is known about the size restrictions that exist within the siRNA binding site of RISC. The long-term objective of this project is to investigate the steric tolerance at various positions of nucleobase-expanded siRNAs bound to RISC in cells. The first aim of this project is to synthesize four novel, benzo-fused RNA (xRNA) nucleosides using methods employed by our laboratory. Since it is known that sequence recognition is important in determining the efficacy of RNAi, the next aim is to use automated synthesis to create antisense strands with single xRNA substitutions at various positions and an unmodified sense strand with an RNA sequence corresponding to Renilla luciferase mRNA. These strands will then be combined to form an siRNA duplex whose biophysical properties (e.g. thermodynamic stability, serum stability and fluorescence) can be characterized. In accordance with the mission statement of PPBC division of the NIGMS to support fundamental science that helps generate a knowledge base for the advancement of diagnostics and therapeutics, it is expected that the results obtained from this study will reveal certain positions on the siRNA strand where steric effects in RISC are most important. This will lead to more practical applications in the design of enhanced siRNAs for biological applications. Thus, the final aim of this project will be to introduce these modified siRNA duplexes into HeLa cells co-transfected with Renilla and firefly luciferase vectors to evaluate the silencing effectiveness of the siRNAs upon the Renilla luciferase gene using a dual Renilla/firefly luciferase reporter assay. This project aims to use modified RNA to study the flexibility of an important set of proteins involved in a recently discovered gene deactivation process found in cells called RNA interference (RNAi). RNAi technology has the potential of treating numerous diseases with the use of specific RNA. Since little is currently known about this set of proteins which drives RNAi, the findings from this work will contribute to the design of more potent and efficient RNAs for therapeutic purposes.